MTC Flashcards
use of cryo-based methods
rapid microscopic analysis
rapid diagnosis
oncologic sx
reasons to perform cryo-based methods
Provide quick gross or microscopic diagnostics to identify an unknown
pathologic process, determine disease extent/margins, detect
metastases, or simply identify a tissue.
* Process tissue to give appropriate and accurate diagnosis and
prognosis, as well as to follow research and particular study protocols.
* Confirm the presence of diseased tissue on permanent sections for
diagnosis.
Reasons not to perform cryo-based methods
The diagnosis of a frozen segment has no immediate ramifications for
decision making.
* Tissue is required for permanent processing (it is unusual or small, or
it requires lengthy research to diagnose).
* Frozen sections are known to exhibit severe artifacts that make
appropriate interpretation difficult.
* Tissue is severely calcified and ossified.
* The possibility of a major infection (e.g. HIV, hepatitis B/C, TB)
arrange
- Tissues are placed on dispensing
slide - Wells are frozen with chuck and
over-chuck - Applying embedding medium
- Tissues are placed in embedding
wells - Block is removed for trimming/
staining - Wells are filled with embedding
media
3 1 4 6 2 5
While awaiting the stain line,
prepared slides should be
immediately immersed in formal
alcohol, 95% alcohol (methanol /
ethanol), or formalin; if this step is
skipped, ___ artifacts will occur
drying
use of IHC
tumor
IHC flowchart
deparaffinization
rehydration
Ag retrieval
blocking
primary antibody
secondary antibody
chromogen application
counterstain
For epitopes which may lose antigenicity with heat, may destroy
epitopes and tissue morphology
enzyme digestion
used method For some drugs such as bleomycin, daunomycin and pepleomycin
oxidizing
used to Minimize contamination of sections
detergent
Background staining can be caused by:
- nonspecific antibody binding - more common in polyclonal antibodies, and
- endogenous peroxidase activity - more problematic in tissues with a high
concentration of hematopoietic components, (e.g. bone marrow) - Nonspecific antibody binding can be reduced by:
> Preincubating the secondary antibody with normal serum from the same species or with
a commercially available universal blocking agent (e.g. goat serum).
targets a single epitope; tends to be more specific
monoclonal antibody
can bind many different epitopes; tends to be more sensitive
polyclonal antibody
function of the secondary antibody
visualize the antigen-antibody reaction
How can nonspecific antibody binding be reduced
preincubating the secondary antibody with normal serum from the same species or with a commercially available universal blocking agent
causes of background staining
- nonspecific antibody binding
- endogenous peroxidase activity
goal of fixation
preservation
most common type of fixation
immersion
selection of fixative should be based on
- prevent degeneration and autolysis
- can harden to enable cutting
- does not distort the cellular constituents
- can support chemicals used in processing
optimum size and thickness for trimming
size: 2x2x0.3cm
thickness: 3-4 mm
Aim of tissue processing
embed the tissue in a solid medium firm enough to support the tissue and give it sufficient rigidity to enable thin sections to be cut
stages of tissue processing
- dehydration
- clearing
- impregnation
- embedding
Standard / best example for clearing medium
Xylene
best example for dehydrating fluid/ agent
ethanol
Preferred embedding medium
paraffin wax
melting point of paraffin wax
52-56 degrees celsius
plastic point (point of solidification)
10 degrees below melting point
precision instrument that cuts sections from the paraffin blocks, thin enough for examination under microscope
microtome
basic principles of staining
- deparaffinization
- hydration
- washing and rinsing
Routine staining
hematoxylin and eosin stain
what does the following stain in an H&E stain
nuclei
cytoplasm
muscle fiber
RBC
calcium
mucin
nuclei - blue
cytoplasm - pink/purple
muscle fiber - deep red
RBC - orange-red
calcium - dark blue
mucin - gray-blue
Van Gieson stain differentiates ___ and ___
collagen and other connective tissues
Van Gieosin stain: collagen: ___, muscle fiber and blood cells: ___
collagen: red
muscle fiber and blood cells: yellow
toluidine blue is attracted to ______
nucleic acids
toluidine blue: nucleic acid:_____, mucin & cartilage: _______
nucleic acid: blue
mucin & cartilage: purple
Masson’s trichrome stain:
nuclei:
cytoplasm, muscle, erythrocyte:
collagen:
nuclei: black
cytoplasm, muscle, erythrocyte: red
collagen: blue/green
PAS (Periodic Acid Schiff) differentiates ____. PAS positive:___
nuclei:
glycogen
PAS positive: pink or red
nuclei: blue
